There are two distinct populations of circulating endothelial cells: bone marrow derived circulating endothelial progenitors (CEPs) and mature circulating endothelial cells (mCECs) (Beaudry et al., 2005, Clin Cancer Res 11:3514; For additional background see Rosenzweig, 2005; New Engl J Med 353:1055-1057; Khan SS, 2004; Cytometry Part B, Clinical Cytometry 64B: 1-8). Measurement of circulating endothelial cells can act as surrogate markers for biological activity of antiangiogenic agents and used to select patients who will benefit from such therapy for cancer (Beaudry et al., 2005, Clin Cancer Res.). For example, patients with CEPs expressing high levels of VEGFR are excellent candidates for treatment with VEGFR inhibitors such as bevacizumab (Avastin). Also, measurement of mCECs is useful in cancer since an increased level indicates disease progression (Mancuso P et al., 2001, Blood 97:3658-61; Beerepoot L V et al, 2004, Ann Oncol 15:139-45).
But “The clinical testing of these [antiangiogenic] agents is currently hampered, by the lack of surrogate markers for measuring their biological effect and predicting which patients are most likely to benefit.” (Davis D W, et al., 2003, Br J Cancer 89:8-14). Therefore there is a need for a test to predict which cancer patients will likely benefit from antiangiogenic therapy.
Circulating endothelial cells are an important prognostic factor in cardiovascular field of medicine. Low levels of CEPs indicate a high risk of cardiovascular events (Hill J M et al, 2003, New Engl J Med 348:593-600; Werner N, et al., 2005, New Engl J Med 353:999-1007; Schmidt-Lucke C et al., Circulation 2005, 111:2981-87). There is a need for a test to better predict which patients are at risk for cardiovascular events. In a patient suspected of having a myocardial infarction (MI), high levels of CEPs suggest the recent occurrence of an MI. There is a need for a better test to predict which patients have had a myocardial infarction if they are suspected of having had one.
The approaches used in the published literature to quantify CEPs and mCECs often use flow cytometry (Werner N et al., 2005, N Engl J Med 353:999-1007) which is described as a “difficult undertaking” (Khan SS et al., 2004). This is a cumbersome and time-consuming approach not readily amenable to automation. A further problem with flow cytometry et al is the high background staining in flow cytometry. Nonspecific staining of 0.1 to 0.5% of cells analyzed is commonly seen with flow cytometry and this is too high and will mask the detection and numeration of the desired circulating endothelial cell population which can be as low as 0.0001% (Khan SS et al. (2004; Cytometry Part B, Clinical Cytometry 64B: 1-8)). Excessive data storage capacity is another problem with rare event analysis by flow cytometry (Khan et al., 2004). Another method used in the literature to measure CEPs is by colony counting (Hill J M et al., 2003, New Engl J Med 348:593-600). This method is slower than flow cytometry taking more than one week to perform (Hill et al., 2003). The method of this invention therefore is a significant advance in the field in that it allows for a rapid assessment of circulating endothelial cells and avoids the difficulties (background issues, excessive data storage capacity) inherent in a rare event analysis by flow cytometry.
CEPs and mCECs represent a very small fraction of mononuclear cells in the blood with an estimate of between 0.01% and 0.0001% (Khan SS et al., 2004). Enumeration of these cells in the peripheral blood is important medically because they provide an insight into the body's angiogenic and neovasculogenic activities. Angiogenesis has been shown to be critical for tumor growth and novel anti-angiogenic therapies (such as the approved agent bevacizumab) are used to slow or prevent tumor growth. Angiogenesis and neovascularization are also detrimental in many other diseases besides cancer, including sickle cell disease, vasculitidis, and pulmonary hypertension (Khan SS et al., 2004). In contrast, in coronary artery disease, neovascularization or revascularization is desirable in order to improve blood flow to the cardiac tissue.